Cytosine deaminase negative selection system for gene transfer techniques and therapies

ABSTRACT

The present invention relates to a system comprising a modified bacterial gene for cytosine deaminase that has been engineered into a eukaryotic expression vector and the expression of the gene by mammalian cells. 
     The present invention further relates to methods, gene therapies and vaccines that employ the negative selectable marker, cytosine deaminase, which has the ability to produce a toxic antimetabolic 5-fluorouracil from 5-fluorocytosine.

This is a division of application Ser. No. 07/725,076 filed Jul. 3,1991, now U.S. Pat. No. 5,358,866.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a system comprising a modifiedbacterial gene for cytosine deaminase that has been engineered into aeukaryotic expression vector and the expression of the gene by mammaliancells.

The present invention further relates to methods, gene therapies andvaccines that employ the negative selectable marker, cytosine deaminase,which has the ability to produce a toxic antimetabolite 5-fluorouracilfrom 5-fluorocytosine.

2. Background Information

Selectable genetic markers are important tools in the study of theregulation and function of genes and are potentially important in genetransfer therapies. Conferring unique resistance or sensitivity tocytotoxic agents enables the skilled artisan the ability to select ordelete genetically altered cells from a mixed population.

The enzyme cytosine deaminase (CD) is useful in the present invention asa selectable genetic marker because of its ability to catalyze thedeamination of cytosine to uracil (M. Kilstrup et al., J. Bacteriology171:2127-2127 (1989); L. Anderson et al., Arch. Microbiol. 152:115-118(1989)). Bacteria and fungi which express this gene convert5-fluorocytosine (5FC) to 5-fluorouracil (5FU) and this metabolite istoxic to the microorganism (A. Polak and H. J. Scholer, Chemotherapy(Basel) 21:113-130 (1975)). Mammalian cells do not express significantamounts of cytosine deaminase and do not deaminate 5FC (A. Polak et al.,Chemotherapy 22:137-153 (1976); B. A. Koechlin et al., BiochemicalPharmacology 15:434-446 (1966)); 5FC is relatively nontoxic to them (J.E. Bennett, in Goodman and Gilman: the Pharmacological Basis ofTherapeutics. 8th ed., eds. A. G. Gilman, T. Rall, A. S. Nies and P.Taylor (Pergamon Press, New York) pp. 1165-1181). However, 5FU haspotent cytotoxic effects on mammalian cells. 5FU is subsequentlymetabolized to FUTP and FdUMP and thereby inhibits both RNA and DNAsynthesis and kills the cell (P. Calabrisi and B. A. Chabner in Goodmanand Gilman: the Pharmacological Basis of Therapeutics. 8th ed., eds. A.G. Gilman, T. Rall, A. S. Nies and P. Taylor (Pergamin Press, New York)pp. 1209-1263); L. E. Damon et al., Pharmac. Ther. 43:155-189 (1989)).Thus, intracellular metabolic conversion of 5FC to 5FU should be lethalto mammalian cells.

The bacterial gene for cytosine deaminase has recently been isolated andcloned (L. Anderson et al., (1989)). The present invention provides anew negative selectable marker in which the gene for cytosine deaminasefrom a microorganism, of which bacteria is an example, is modified andintegrated into a eukaryotic expression vector and expressed inmammalian cells conferring upon the transfected cells a uniquesusceptibility to the cytotoxic effects of 5FC. The present inventionalso provides methods that use the cytosine deaminase negative selectionsystem in vitro to selectively eliminate subpopulations of cells, and invivo for gene transfer therapies and vaccines.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel expressiongene construct containing a modified cytosine deaminase (CD) gene inmammalian cells and subsequent sensitivity of the mammalian cellsengineered with the modified CD gene to 5-fluorocytosine (5FC).

Another object of the present invention is to provide methods thatrequire application of the CD gene construct or modifications of it in avariety of therapeutics including immunotherapy, gene therapy and bonemarrow transplantation.

Various other objects and advantages of the present invention willbecome obvious from the drawings and the detailed description of theinvention.

In one embodiment, the present invention relates to a DNA construct thatcomprises a modified cytosine deaminase gene from a microorganism and aeukaryotic expression vector. Examples of microorganism are bacteria andfungi.

In another embodiment, the present invention relates to the DNA plasmidconstruct designated pCD2 which has the accession #40999.

In a further embodiment, the present invention relates to a mammalianhost cell comprising the DNA construct of the modified cytosinedeaminase gene and a eukaryotic expression vector. In a further aspectof the invention, mammalian host cells comprising the modified bacterialcytosine deaminase DNA construct expresses cytosine deaminase protein.

In another embodiment, the present invention relates to a CD negativeselection marker system that provides a safety system in gene transfertherapies comprising the steps of inserting the DNA construct comprisinga modified CD gene, eukaryotic expression, and exogenous DNA into a hostor patient genome and treating the host or patient cells with 5FC inpharmacologically acceptable doses that will selectively kill cells thathave integrated the DNA construct into their genome.

In another embodiment, the present invention relates to a method of genetherapy that regulates the gene product expression in a host comprisingthe steps of inserting a DNA construct comprising a modified CD gene,eukaryotic expression vector, and a therapeutic gene of interest into ahost cell resulting in altering the host cell and treating the alteredhost cell with periodic does of 5FC in pharmaceutical amounts such thatthe numbers of the host cell is diminished but not completely destroyed.In a variation of the method of regulating gene product expression in ahost the methods described above is modified such that treatment with5FC is in higher doses such that all the altered cells are destroyed.

The present invention further relates to live tumor vaccines for mammalscomprising the modified CD gene and eukaryotic expression vector andtumor cells.

In another aspect, the present invention provides a method for treatingtumors in a patient comprising the administration of acceptable doses oflive the vaccine described above to a patient and subsequentlyadministering a high dose of 5FC that will destroy the live cells usedas vaccine.

In a further embodiment, the present invention relates to a vaccine formammals against a microbiologic pathogen comprising a live unattenuatedvirus, bacteria or protozoa, a modified CD gene and expression vector inamounts sufficient to induce immunization against the virus, bacteria orprotozoan.

In a further embodiment, the present invention relates to a method ofvaccination against a microbiological pathogen comprising administeringthe vaccine described above to a host and subsequently administering ahigh does of 5FC sufficient enough to destroy the live immunogen.

In a further embodiment, the present invention relates to a method ofadministering an allogeneic or autologous bone marrow transplant into apatient comprising the steps of treating the bone marrow transplant witha modified CD construct packaged into a vector in such a matter that theconstruct will preferentially infect tumor cells or lymphocytes but notbone marrow stem cells and subsequently treating the bone marrow cellswith 5FC in doses such that the tumor cells or lymphocytes arecompletely purged or destroyed and administering the treated bone marrowcells in patient. In a modification of the above technique, the 5FCtreatment may be given subsequent to the administration of the bonemarrow transplant in a patient.

In yet another embodiment, the present invention relates to a doublenegative selection vector comprising the modified CD gene, and theherpes thymidine kinase gene in a eukaryotic expression vector.

In yet another embodiment, the present invention relates to a diagnosticmethod for detecting successful homologous recombination eventscomprising the steps of inserting a modified CD DNA construct into acell line in vitro, creating a deletion mutant that will retainsignificant homology in a DNA sequence of the CD DNA construct butrender the CD gene biologically inactive and detecting successfulhomologous recombination by measuring the loss of sensitivity of 5FC.

In another embodiment, the present invention relates to a method forselectively eliminating tissues in an animal comprising the steps ofinserting a modified CD DNA construct comprising a modified CD gene anda tissue specific promoter into an animal cell and subsequently treatingthe animal with 5FC to eliminate the tissue corresponding to the tissuespecific promoter.

In yet another embodiment, the present invention relates to a method ofcancer therapy in a patient comprising the steps of administering to apatient a therapeutic dose of a DNA construct comprising a modified CDgene and a promoter with a predilection for transducing cancer cells andsubsequently treating the patient with a toxic dose subsequentlytreating the patient with a toxic does of 5FC that will destroy thecancer cells but not other cells.

The entire contents of all publications mentioned herein are herebyincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the construction of pCD2. Cloning of the cytosine deaminasegene into eukaryotic expression vector pLXSN was performed in thefollowing way. The 1.7 kb cytosine deaminase fragment from pMK116digested by HincII and BamHI was ligated into the polycloning site ofpLXSN digested with HpaI and BamHI, producing pCD1. The sequence ofpCDp1 in the 5' region at the EcoRI site near the start site GTG isshown. Oligonucleotide directed mutagenesis of pCD1 and recloning of themodified cytosine deaminase gene into pLXSN to produce pCD2 weresubsequently undertaken. PCR primers SEQ ID NOS.: 1 & 2, (FIG. 1) andpMK116 (L. Anderson et al., (1989)) were used to eliminate upstreamATG's and change GTG to ATG at the start codon. The modified cytosinedeaminase gene was then cloned into pLXSN. The sequence of pCD2 in the5' region of the start codon beginning at the EcoRI site near is shown(SEQ ID NOS.: 3 & 4).

FIGS. 2A-B is a Southern analysis detecting the presence of cytosinedeaminase gene in genomic DNA of cells transfected with pCD2.

Panel A represents the Southern analysis on PCR products using cellularDNA as template for cytosine deaminase DNA synthesis. 1 μg cellular DNAunderwent PCR using primers corresponding to the 5' and 3' ends of themodified cytosine deaminase gene. PCR product was electrophoresed,blotted by capillary transfer and probed with a 32P-labelled cytosinedeaminase probe. The bands seen correspond to a 1.7 kb fragment.

Panel B represent the Southern analysis of cellular DNA. 10 μg cellularDNA was digested with SacI, electrophoresed, transferred and probed withthe same probe. SacI cuts through both LTR elements of pCD2 and shouldgenerate from unrearranged DNA a 4.5 kb fragment containing the cytosinedeaminase sequence. As a positive control 1 μg 3T3 DNA was supplementedwith 30 pg pCD2 (3T3+pCD2) prior to digestion with Sac I.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in part, to the insertion of the acytosine deaminase (CD) gene derived from a microorganism into aeukaryotic expression vector. A principle embodiment of this aspect ofthe present invention relates to the successful expression of the CDgene in mammalian cells and the subsequent sensitivity of cellsexpressing the gene to the toxic effect of 5-fluorocytosine, an agentnontoxic to unaltered mammalian cells. The present invention alsorelates to methods that apply to the above CD gene selectable marker ingene transfer studies and therapies.

The cytosine deaminase (CD) gene underwent modification in and aroundthe start site for eukaryotic expression. Without these modificationsthe cytosine deaminase was poorly expressed in mammalian cells even whencloned into a strong expression vector like pLXSN. In particular, thepresent invention relates to the constructed bacterial gene for CD in aneukaryotic expression vector, for example, pCD2 and the expression ofthe gene in mammalian cells, for example, murine fibroblasts.

The cytosine deaminase gene is found and expressed in a variety ofmicroorganisms. Examples include the fungi Cryptococcus neoformans,Candida albicans, Torulopsis glabrata, Sporothrix schenckii,Aspergillus, Cladosporium, and Phialophora (J. E. Bennett, Chapter 50:Antifungal Agents, in Goodman and Gilman's the Pharmacological Basis ofTherapeutics 8th ed., AG Gilman, ed., Pergamon Press, New York, 1990)and the bacteria Escherichia coli and Salmonella typhimurium (L.Andersen, et al., Archives of Microbiology, 152; 115-118, 1989). Inthese microorganisms the genetically encoded enzyme serves the samepurpose: to help provide uracil from cytosine for nucleic acidsynthesis. The E. coli enzyme and gene are representative of the group.

The distinguishing feature of the present invention is the expression ofthe CD gene in higher eukaryotic cells for the purpose of creating anegative selection system. One skilled in the art of molecular biologymay express the modified CD gene in a variety of other eukaryoticexpression vectors to achieve the same purposes as those disclosedherein.

Introduction of the gene into mammalian cells results in the ability oftransfected cells to convert cytosine to uracil; normally mammaliancells do not contain the enzyme cytosine deaminase. In vitro conversionof radiolabelled cytosine to uracil is consistently seen withtransfected cells. The presence and expression of the gene has noapparent deleterious effects upon the cells if they are not exposed to5FC. However, when such cells were exposed to 5FC they ceaseproliferation and die as judged by their failure to produce colonies inclonogenic assays and impaired proliferation in ³ H-thymidine uptakeassays (See Example 3). The toxicity was due to the deamination of 5FCto 5FU by the cells. Normal cells were not inhibited by 5FC and onlythose cell lines with demonstrable cytoside deaminase activity in vitrowere sensitive to 5FC toxicity.

The ability to render cells selectively susceptible to the toxic effectsof 5FC is important in implementing the present invention of the CDnegative selection system in a variety of therapeutic assays andvaccines described below.

The present invention describes the novel expression of the modifiedcytosine deaminase gene in mammalian cells and subsequent sensitivity ofcells engineered with the modified CD gene to 5FC. The inventionsdescribed below involve either direct application of the plasmid pCD2 ormodifications of it that would be easily performed by an individualskilled in the art of molecular biology given the information applicantshave provided about the cytosine deaminase negative selection system andknowledge and materials regarding other eukaryotic gene expressionpromoters/enhancers and retroviral packaging cell lines that are in thepublic domain.

The present invention is the first to demonstrate that the bacterialenzyme for cytosine deaminase when appropriately modified in sequenceand placed under control of a eukaryotic promoter can be inserted intothe genome by transfection or retroviral transduction and render cellsselectively sensitive to 5FC. By providing the present invention ofrendering mammalian cells sensitive to 5FC through engineering of thecytosine deaminase gene, one skilled in the art will be enabled to applythe cytosine deaminase negative selection system (CDNSS) to a variety oftissues by simply applying known techniques in molecular biology andretrovirology. Multiple tissue-specific promoter/enhancer sequences havebeen described. As representative examples of these promoter/enhancerelements the following references are listed: Muscle and neural: E.Barnea et al. Neuron 5:881-888 (1990); Thyroid: C. Ledent et al., Proc.Natl. Acad. Sci. U.S.A. 87:6176-6180(1990); Lymphoid: G. P. Cook & M. S.Neuberger, Nucleic Acids Res. 18:3665-3671 (1990); hepatic P. Herbomelet al., Mol. Biol. 9:4750-4758 (1989) and M. G. Izban et al., J. Biol.Chem. 264-9171-9199 (1989); Bone Marrow: J. Magram et al., Mol. CellBiol. 9:4750-4758 (1989).

Using cloning techniques described herein and elsewhere atissue-specific promoter/enhancer could be inserted in pCD2 resulting inactivation of the cytosine deaminase gene in a specific tissue. Oneexample of this would be use of the immunoglobulin heavy chainpromoter/enhancer to activate the gene in B-cells (S. Eccles et al. NewBiol. 2:801-811 (1990); J. Wang et al., Mol. Cell Biol. 11:75-83 (1991);B. Porton et al. Mol. Cell Biol. 10:1076-83 (1990); C. Queen & D.Baltimore, Cell 33:717-728 (1983); E. E. Max. "Immunoglobulins:molecular Genetics" in W. E. Paul (ed.) Fundamental Immunology (2nd.ed.) Raven Press, N.Y., pp. 235-290 (1989)).

Another way to confer tissue specificity may be to deliver the CDNSSusing the pCD2 plasmid in different packaging cell lines, an example ofwhich is the cell line PA317 we describe here. A variety of retroviruspackaging lines which have different cell-type and species tropisms havebeen described (A. D. Miller Human Gene Therapy 1:5-14 (1990)). Oneskilled in the art of molecular biology would have no difficulty takingthe invention and transfecting it by calcium phosphate precipitationinto a different packaging cell line which would then change the targetcell specificity of the CDNSS.

In one embodiment, the present invention relates to a CD negativeselection marker system that may provide a safety system in genetransfer therapies. Because gene therapy involves insertion of exogenousDNA into a host's or patient's genome, it is possible that malignanttransformation of the target cells will result. The CD system may beused to destroy the malignant cells.

This application could be achieved in several ways given the presentinvention described herein about the cytosine deaminase negativeselection system (CDNSS). First, target cells for gene therapy couldfirst be treated with CDNSS and only cells that have incorporated thesequence as judged by neomycin resistance could then be subject to genetherapy with a second vector carrying a therapeutic gene. This two-stepprocess would ensure than any cells altered by the therapeutic genevector would also have the CDNSS present.

Second, the CDNSS could be modified by someone skilled in the art ofmolecular biology to incorporate the CDNSS and the therapeutic gene inthe same vector. The number of ways of accomplishing this is great. Oneexample would be to clone a therapeutic gent and a promoter element intopCD2, either replacing the neomycin resistance gene or by adding a thirdgene to pCD2. Another example would be excision with restrictionendonucleases of the unique element of the CDNSS, the modified cytosinedeaminase gene, and its cloning into another plasmid or retrovirus thatharbors the therapeutic gene. Any cell altered by the vector would thencontain the CD gene. If that cell or its progeny became malignant, thepatient or host could be treated with 5FC (5-fluorocytosine) and thecells would be killed.

Similarly, the CD gene could be used to destroy cells altered by genetherapy if they produced a substance that was toxic to the patient. Forexample, if tumor necrosis factor was to be used in gene therapy, theCDNSS of modified cytosine deaminase gene could be included in thevector. Then if the transferred cells made amounts of TNF that weretoxic to the host, the patient could receive 5FC. The transferred cellswould be destroyed and the TNF production would cease. This approach maybe used with any therapeutic gene that may have unforeseen orunacceptable side-effects.

The present invention further relates to a method for controlling thegene expression in gene therapy by applying the CD negative selectionsystem. The CD system of the present invention may be used to regulatethe amount of gene product the host or patient receives.

The CDNSS could be used to regulate gene dosage in a manner similar tothat described above in the discussion of controlling malignant cells orcells that were producing unacceptable side-effects. That is, targetcells could first be engineered with the unmodified CDNSS and only cellsthat had first been modified with it could then be subjected to a secondmanipulation with a vector containing a therapeutic gene. Or anindividual skilled in the art of molecular biology could move theessential element of the CDNSS, the modified CD gene, into anothervector that harbored the therapeutic gene and use a single vector totransfer the CDNSS and the therapeutic gene. An example of theapplication follows. For example, a gene for erythropoietin and CD maybe cloned into the same vector used in gene therapy to treat anemia.After infusion of many vector containing cells, a high serum level ofhormone may be achieved. Low doses of 5FC could be given periodically tothe patient to diminish in size the pool of cells producing hormone andthus reduce the serum level of the hormone to the level desired.Similarly, if one wished to expose a patient to gene therapy for onlyshort periods of time instead of permanently keeping gene altered cellsin the patient, high doses of 5FC could be given to the patient at theend of the prescribed period and destroy all the producing cells. Thismethod may be used with any therapeutic gene, for example, insulin,growth hormone, clotting factors or growth factor.

The present invention further relates to live tumor vaccines containingthe CD gene. Immunotherapy of malignancy may involve using a vaccine ofa host's tumor extracts or cells to enhance the host's immune responseto the tumor (B. Gansbacher et al., J. Exp. Med. 172:1217-1224 (1990)).Live vaccines are more effective than killed cells or cell extracts ashas been shown by vaccine development in the areas of virology andbacteriology. However, administering live tumor as vaccine to patientsis dangerous because the early immune response to it may not completedestroy it; the tumor still would have the potential to invade locallyand metastasize. However, introducing the CDNSS into live tumor cells bytransfection, by retroviral transduction or other gene transfertechniques using the same methods used with 3T3 or PA317 cells wouldthen make the tumor sensitive to 5FC. This may allow safe use of a livetumor vaccine. For example, two weeks after inoculation of tumor thepatient or host could receive 5FC. This would destroy the tumor inoculumbut leave the immune cells of the host undamaged.

The present invention further relates to novel live vaccines and novelmethods for producing "attenuated" or controllable pathogens asimmunogens. Some viruses, bacteria and protozoa are quite virulent andcannot be used in immunization. Traditional methods of developingattenuated strain can result in organisms that are not optimallyimmunogenic. The CD system of the present invention may be used toproduce a controllable pathogen for immunization. The unique feature ofthe CDNSS in this context is its ability to destroy with 5FC cells thatharbor intracellular pathogens. Using the techniques described herein toclone the modified cytosine deaminase sequence gene into pLXSN, theCDNSS could be modified to contain the cytosine deaminase gene andelements of other viruses. For example, elements of the HIV genome (theagent responsible for AIDS and for which techniques of live virusimmunization are currently unsafe) could be cloned into the CDNSS. Themodified gene expression system would express cytosine deaminase andHIV. The modified CDNSS could then be given to patients and after theinnoculation had initiated an immune response to the HIV elements 5FCcould be administered to and inhibit further transcription andtranslation of the HIV elements. This would be analogous to treatment ofherpes simplex infections with acyclovir or ganciclovir (R. G. Douglas,"Antiviral Agents" In A. G. Gilman (ed) Goodman and Gilman's thePharmacological Basis of Therapeutics (8th ed), pp. 1184-1887 (1990)).

In yet another embodiment, the present invention relates to a method oftherapy of human immunodeficiency virus (HIV) infection using the CDnegative selection system. As noted in other parts of this application,the CDNSS could be altered by someone skilled in the art of molecularbiology in a variety of ways. One alteration would be the replacement ofthe promoter driving expression of the cytosine deaminase gene withanother promotor. Promotor/enhancer elements from the HIV genomeresponsive to HIV transactivation could be inserted upstream of thecytosine deaminase gene (KA Jones New Biol. 1:127-135 (1989)).Intracellular HIV activity would then result in activation of thecytosine deaminase gene. This would provide a novel therapy for HIVinfection. White blood cells from an HIV positive individual could beremoved by standard leukapheresis, infected in vitro with the CDNSS andreturned to the patient who would then receive 5FC. Cells that containedHIV would then activate the CDNSS via transactivation and be eliminatedby 5FC. The propagation of the HIV infection would thus be curtailed inthe patient.

In another embodiment, the present invention relates to a therapeuticmethod for use in allogeneic or autologous bone marrow transplantation.It is often desirable in bone marrow transplantation to eliminatecertain cells from the bone marrow before they are infused into apatient. For example, one may want to purge residual tumor cells oreliminate certain cells that could cause graft-versus-host disease inthe bone marrow recipient. The CD system of the present invention may beused in such purging strategies. For example, it could be packaged intoa vector that will preferentially infect tumor cells but not the bonemarrow stem cells. The tumor cells but not the stem cells will besensitive to 5FC.

It has been documented that retroviral gene insertion will not occur incells that are not replicating, i.e., those that remain in G₀ of thecell cycle (D. G. Miller, et al. Mol. Cell Biol. 9:1426-1434 (1990)).This phenomenon provides a basis for selective infection of tumor cellsin bone marrow infiltrated with tumor. The bone marrow stem cellswithout specific hormonal stimulation in vitro remain quiescent whilethe tumor cells naturally cycle. Exposure of this infiltrated marrow toretrovirus carrying the CDNSS will then result in insertion of the CDgene into the tumor cells but not the quiescent bone marrow stem cells.Pretreatment of the bone marrow prior to infusion or administration of5FC to the patient after bone marrow infusion will result in purging ofthe infected tumor cells. Similarly, the CD gene of the presentinvention may be packaged in a vector that will preferentially infectlumphocytes but not stem cells and 5FC used to purge the marrow oflymphocytes prior to infusion, or administered to the patient to preventor treat graft-versus-host disease.

In a further embodiment, as a diagnostic assay, the CD system of thepresent invention may be used as a reporter marker for successfulhomologous recombination. Using methods identical to those described toinsert the CDNSS system into the 3T3 or PA317 cells, one could stablyintegrate the CDNSS into a cell line in vitro. One skilled in the art ofmolecular biology could use the present invention regarding therestriction sites in the CDNSS to create a deletion mutant which wouldretain considerable homology to the DNA sequence of the CDNSS but wouldnot yield biological active cytosine deaminase. This technique iscommonly used in molecular biology. The deletion mutant and the CDNSScould then be used in homologous recombination trials with loss ofsensitivity to 5FC as a marker for successful recombination. Suchhomologous recombination trials have been performed with otherselectable markers or reporter genes in studies of targeted geneinsertion (R. J. Bollaf, et al., Annu. Rev. Genet. 23:199-225 (1989)).

In another embodiment, the CD system of the present invention may beused in co-cultivation transduction methods with viral vectors.

A known technique in molecular biology and retroviral gene transferstudies is cocultivation in vitro of target cells and the retrovirusproducer cell line. This results in intimate contact of target cellswith supernatant whose retrovirus content is continuously being renewed(M. A. Eglitis, et al., Science 230:1395-1398 (1985); M. A. Eglitis & W.F. Anderson, BioTechniques 6:608-614 (1988)). In viral transductiontechniques of gene transfer, cell-free viral supernatant is harvestedfrom virus producer cell lines that produce virus. Target cells are thenexposed to the supernatant. However, the viral supernatant is unstableat temperatures used for transduction and loses all its activity in afew hours. An alternate strategy is to mix in culture live virusproducer lines and target cells. They can be cocultivated for a longtime resulting in much more efficient transduction of the target cells.This is because the producer line is continuously making live virus.This cannot be done for gene therapy currently because it is verydifficult to separate the producer and target cell lines and have onlypurified target cells to give to the patient. However, if the CD genesystem of the present invention were transfected into the producer linein a form that would not result in packaging of the CD gene, the virusproducer cells could be purged from the co-cultivation culture by adding5FC to the medium after the desired period of virus exposure wascomplete. Then only the desired target cells would survive and may thenbe given to the host.

The present invention further relates to novel methods that createdouble negative selection vectors. The CD gene system of the presentinvention may be inserted along with the herpes thymidine kinase geneinto a gene transfer vector along with other genes. Then the cellsreceiving the vector may be sensitive to both 5FC and ganciclovir oracyclovir, providing a double negative selection system for eliminatinggene modified cells. This may be advantageous as some cells may not beeliminated with CD/5FC or TK/ganciclovir alone (F. L Moolten & J. M.Wells, Journal of the Natl. Cancer Inst. 82:297-300 (1990)). Incombination they may provide additive or possibly even synergistictoxicity.

The present invention also relates to methods for producing transgenicanimals by incorporating the CD gene of the present invention into thegerm-line of an animal. The CD gene system of the present invention maybe inserted into the germ-line of an animal, for example, a mouse. TheCD gene may be combined with a variety of tissue-specific promoters,which will result in CD being expressed only in those tissues in whichthe promoter is active, for example, in B-cells if an immunoglobulinpromoter is used (E. Borrelli et al., Proc. Natl. Acad. Sci. U.S.A.85:7572-7576 (1988)). The 5FC may be used to selectively eliminate thesetissues. This will be of use in studies of organ and tissue development.

In a further embodiment, the present invention relates to a therapeuticmethod for the treatment of cancer. As currently configured the CDNSS ofthe present invention will have a predilection for transduction ofcancerous tissues as opposed to normal, nonneoplastic tissue. Asdiscussed above, it has been shown that replicating cells permitretrovirus mediated gene insertion but quiescent cells do not (A. D.Miller, 1990). A patient could then be treated in vivo with the CDNSS ofthe present invention in retrovirus form and the patient's cancerouscells would be preferentially tranduced and become sensitive to 5FC. Oneskilled in the art of molecular biology would be able to increase thetissue specificity of the CDNSS by taking the information provided bythe present invention of the CDNSS regarding the ability to render cellsselectively sensitive to 5FC and by inserting known tissue-specificpromoters into the CDNSS using standard techniques in molecular biology.(See earlier discussion of tissue-specific promoters for references).For example, one could rearrange the elements of the CDNSS in thefollowing way to make the CDNSS active in B-cell leukemias andlymphomas. Using standard cloning techniques such as those used in theconstruction of pCD2 one could move the neomycin phosphotransferase geneimmediately 3' to the LTR promoter, insert the promote/enhancer for theimmunoglobulin heavy chain gene 3' to the neomycin phosphotransferasegene and 5' to the modified cytosine deaminase gene, and then delete theSV40 promoter. As the immunoglobulin promoters are preferentially activein cells of the B-lymphocyte lineage (S. Eccles et al. New Biol.2:801-811 (1990); J. Wang et al. Mol. Cell Biol. 11:75-83 (1991); B.Porton et al. Mol. Cell Biol. 10:1076-1083 (1990); C. Queen & D.Baltimore, Cell 33: 717-728 (1983); E. E. Max "Immunoglobulins:Molecular Genetics: in W. E. Paul (ed.) Fundamental Immunology (2nd. ed)Raven Press, N.Y. pp. 235-290 (1989)), the rearranged CDNSS would beuseful in treatment of B-cell leukemias and lymphomas. The specificityfor the tumor cells would be twofold: retroviral preference forproliferating cells for gene transfer and activation of the gene incells of B-lymphocyte lineage.

The invention is described in further detail in the followingnon-limiting examples.

EXAMPLES

The following materials and methods were used throughout the Examples.

Molecular Techniques

The plasmid pMK116 contains a 1.7 kb fragment from E. coli whichcontains the coding region for cytosine deaminase in the polycloningsite of the vector pTZ18U (D. A. Mead et al., Protein Engineering1:67-74 (1986)). The plasmid pLXSN contains eukaryotic expressionelements: (5') Moloney murine sarcoma virus LTR promoter, polycloningsite, SV40 early promoter, neomycin phosphotransferase gene, and Moloneymurine leukemia virus promoter (3') (A. D. Miller and G. J. Rosman,BioTechniques 7:980-990 (1989)). The neomycin phosphotransferase geneallows cells to survive in the presence of the protein synthesisinhibitor neomycin or its analogue G418 (F. Colbere-Garapin et al., J.Mol. Biol. 150:1-14 (1981)). These vectors and the subsequent constructsare depicted in FIG. 1. pMK116 was digested with the restriction enzymesHincII and BamHI. pLXSN was digested with HpaI and BamHI. The 1.7 kbfragment from pMK116 and the 5.7 kb fragment from pLXSN were separatedand isolated by electrophoresis in a low-melting point agarose. Thefragments were then ligated with T4 ligase and transformation competentE. coli were transformed with the product (K. Struhl, BioTechniques3:452-453 (1985)). Minipreps of individual colonies of transformantswere screened for insertion and proper orientation of the cytosinedeaminase gene in pLXSN by restriction digest analysis. Large scalepreparation of plasmids were produced by standard methods and theplasmids purified by cesium chloride gradient centrifugation or byQuiagen columns (J. Sambrook et al., Molecular Cloning: A laboratorymanual. 2nd ed. (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)). pCD1 represented the insertion of the unmodifiedbacterial cytosine deaminase sequence into pLXSN.

Oligonucleotide Directed Mutagenesis of pCD1

Oligonucleotides were synthesized on an Applied Biosystems 381A DNASynthesizer and purified by polyacrylamide gel electrophoresis (J.Sambrook et al., (1989)). Oligonucleotides 5'TGA CGC GAA TTC AGG CTA GCAATG TCG 3' (corresponding to the 5' end of the cytosine deaminasesequence) and 5'CAC ACA TTC CAC AGC GGATCC3' (antisense to the 3' regionflanking the gene) were used as primers and pCD1 was used as template ina polymerase chain reaction using a Perkin Elmer Cetus DNA thermalcycler. The resulting 1.7 kb fragment and pLXSN were digested with EcoRIand BamHI, electrophoretically isolated and ligated with T4 ligase. E.coli were transformed and plasmids screened as above. The resultingplasmid with the altered cytosine deaminase sequence is called pCD2. The5' region of the gene was sequenced by the dideoxynucleotide chaintermination method to verify the desired sequence (J. Sambrook et al.,(1989)). The same PCR primers mentioned above were used to amplifycytosine deaminase sequences in 1 μg of purified genomic DNA fromtransfected cells; the PCR product underwent Southern analysis usingstandard techniques (J. Sambrook et al., (1989)) and a ³² P-labelledprobe corresponding to the 1.7 kb cytosine deaminase gene found betweenthe EcoRI and BamHI sites in pCD2 (J. Sambrook et al., (1989)) Southernblots using the same probe were also performed on SacI digests of 10 μgsamples of purified genomic DNA from cell lines.

Cellular Techniques

Cells were grown in D10, i.e., DMEM supplemented with 10% vol.heat-inactivated fetal calf serum, 2 mM glutamine, 50 U/ml penicillinand 50 μg/ml streptomycin, and incubated at 37° and 5% CO₂. NIH-3T3cells and PA317 cells are mouse fibroblast cell lines which have beenpreviously described (A. D. Miller and C. Buttumore, Mol. Cell. Biol.6:2895-2902 (1986)). PA317 has been derived from NIH-3T3 cells andcontains a stably integrated replication incompetent retroviral genome;it functions as a retroviral packaging line when transfected withplasmids containing a sequence encoding a retrovirus with an intactpackaging signal. Plasmids pLXSN, pCD1 and pCD2 contain retroviral LTR'sand an intact packaging signal. Cells were transfected with purifiedplasmid DNA using a standard calcium phosphate precipitation method (J.Sambrook et al., (1989)). The procedure for viral transduction has beenpreviously described (K. Cornetta and W. F. Anderson, J. Virol. Meth.23:186-194 (1988)). 3T3 cells were grown in supernatant from packaginglines and protamine 5 μg/ml for 24 hours and then D10 medium wassubstituted. 72 hours after transfection or 48 hours after transductionG418 1 mg/ml was added to the medium and cells were selected in thismedium for 7 days. Thereafter the cells were maintained in D10 mediumonly. Clonogenic assays were performed as follows. Cells were diluted to10⁴ /ml and 0.1 ml were placed into 4 cm flat-bottomed wells of a 6 wellCostar tissue culture dish along with 5 ml of medium with 5FC an/or G418at concentrations described in the examples below and tables. They wereincubated for 5 days at which time the wells were stained with Geimsastain and colonies of greater than 25 cells were examined and countedwith the aid of a 40X microscope. Proliferation assays were performed asfollows. 10³ cells were placed in flat bottomed wells of a 96 wellplates containing 0.2 ml medium with additives as described in thetables and text. At the time indicated the wells were pulsed with 25 μl3H-thymidine in RPMI with an activity of 0.5 μCi/25 μl. They wereharvested 4 hours later with an automated cell harvester and counted ina scintillation counter. 12 replicates of each condition were performed.Standard t-test statistical methods were employed (G. W. Snedecor and W.G. Cochran, Statistical Methods ed. 7. Iowa State University press,Ames, Iowa pp. 89-98, 124-128 (1980)).

Enzyme Assay

In vitro assay for cytosine deaminase was performed using a modificationof previously described methods (L. Anderson et al., (1989)). 1×10⁶cells were centrifuged in a microfuge, washed once in normal saline,centrifuged again and resuspended in 10 μl of 100 mM Tris pH 7.8, 1 mMEDTA and 1 mM dithiothreitol. They were then subjected to 5 cycles ofrapid freezing and thawing. The material was centrifuged 5 min. in atabletop microfuge. 10 μl of supernatant was combined with 10 μl3H-cytosine (5 mM cytosine in 100 mM Tris pH 7.8 with an activity of 0.5μCi per 10 μl) and incubated for 4 hours. 10 μl of sample and 10 μl of amarker solution containing unlabelled cytosine 0.4 mg/ml and unlabelleduracil 0.4 mg/ml in water were placed on this layer chromatographysheets (Kodak Chromatogram Sheet 13254) and developed in a mixture of1-butanol (86) and water (14%). After drying, spots corresponding tocytosine and uracil were cut out under short wave UV illumination andassayed in a scintillation counter. The radioactivity recovered from thecytosine and uracil bands accounted for essentially all the labelintroduced to the sample as judged by counting the activity of 10 μl oflabel not subjected to chromatographic separation.

EXAMPLE 1 Rationale for Gene Cloning

FIG. 1 summarizes the cloning process. Initially the entire unmodifiedcoding region for cytosine deaminase from pMK116 was cloned into thepolycloning site of pLXSN. The resulting construct was named pCD1. Whentransfected into 3T3 cells there was little evidence of gene expression.Sequencing of the noncoding region immediately 5' of the coding regionof the cytosine deaminase gene revealed the following sequence:

    __________________________________________________________________________    5' . . . CA ATGTCGC ATGTGGAGGCTAACA GTGTCG . . . 3' (SEQ ID NO.: 5, FIG.      1).                                                                           __________________________________________________________________________

Analysis of the protein in bacteria revealed that translation began atthe GTG codon. As described in the material and method section above andoutlined in FIG. 1, the 5' upstream sequence was altered usingoligonucleotide directed mutagenesis and the gene was cloned into pLXSNunder the LTR promoter upstream of the polycloning site. The resultingplasmid containing the engineered sequence is called pCD2. thisconstruct has been deposited at the American Type Culture Collection inRockville, Md. on Apr. 11, 1991 under the terms of the Budapest Treaty.The plasmid has been given the accession number of 40999. Sequencing ofthe 5' region of the cytosine deaminase gene verified the desiredsequence and deletion of 88 base pairs upstream of the start site inpCD1, the unmodified plasmid containing the bacterial gene. FIG. 1summarizes the salient sequences from pCD1 (the unmodified sequence) andpCD2. pCD2 contains the following eukaryotic expression elements: LTRpromoter promoting the cytosine deaminase gene followed by the SV40early promoter promoting the gene encoding neomycin phosphotransferase.

EXAMPLE 2 Transfection of Mammalian Cells With pCD2 Results inExpression of the Cytosine Deaminase Gene

3T3 and PA317 cells were transfected with pCD2 and 72 hours later wereplaced in medium containing G418 1 mg/ml. The cells were incubated inG418 for 7 days and then maintained in regular medium. Resistance to theneomycin analogue G418 allowed for enrichment of the population of cellswith those that had taken up and incorporated plasmid sequences. Line3T3-CD represents a transfection of 3T3; PA-CD-A and PA-CD-B representseparate transfections of PA317. Incorporation of the cytosine deaminasegene into the genome was demonstrated in two ways. First, PCR reactionsemploying primers corresponding to the 5' and 3' ends of the cytosinedeaminase gene (see FIG. 1) were used to amplify the gene; Southern blotusing a full length cytosine deaminase DNA probe demonstrated the 1.7 kbgene in 3T3-CD, PA-CD-A and PA-CD-B DNA but not in control 3T3 or PA317(FIG. 2). Second, Southern analysis of genomic DNA demonstrated the gene(FIG. 2).

These cell populations was assayed for expression of the cytosinedeaminase gene. An in vitro enzyme assay measured the conversion ofradiolabelled cytosine to uracil by lysates of cells. Cell lines 3T3-CD,PA-CD-A and PA-CD-B demonstrated cytosine to uracil while thenontransfected control lines did not (Table 1).

                  TABLE 1                                                         ______________________________________                                        Conversion of cytosine to uracil in vitro by lysates of                       cell lines containing the cytosine deaminase gene.                                     Cytosine deaminase activity, Units.sup.(1)                           Cell line lysate                                                                         Exp. 1  Exp. 2     Exp. 3                                                                              Exp. 4                                    ______________________________________                                        Buffer only                                                                              0.2     0.1        0.1   0.1                                       3T3        0.6     0.1        0.1   0.1                                       3T3-CD     5.6     1.7        3.4   --                                        PA317      --      0.1        0.1   --                                        PA-CD-A    13.3    17.0       15.7  17.1                                      PA-CD-B    --      18.3       16.1  --                                        3T3-CD-V1  --      --         --    17.5                                      3T3-CD-V2  --      --         --    16.4                                      ______________________________________                                         .sup.(1) Values represent conversion of radiolabeled cytosine to iracil b     cell lysates in vitro. 1 unit of cytosine deaminase activity is defined a     1 U = 1 pmole uracil produced/10.sup.8 cells/min.                             (--) = not done.                                                         

EXAMPLE 3 Cell Lines Expressing the Cytosine Deaminase Gene AreSensitive to 5FC Toxicity

Clonogenic assays were performed to assess the sensitivity of cells to5FC. 10³ cells were inoculated into 4 cm wells and after 5 days thenumber of colonies resulting from the inoculum were counted. Individualcells that can survive and proliferate in that environment can give riseto individual colonies. The inoculum was dilute enough to allow easyidentification and enumeration of individual colonies. One would predictthat cells expressing the cytosine deaminase gene could not give rise tocolonies in the presence of 5FC as they would produce toxic 5FU.Similarly cells not expressing the neomycin resistance gene ought not togrow in the presence of G418.

Assuming a sequence containing both cytosine deaminase and neomycinresistance genes has been integrated into the cell's genome and thatboth genes are expressed, no cell which can survive in G418 shouldsurvive in 5FC and the relative cloning efficiency should drop to nearzero. Table 2 demonstrates that this is indeed the case for those celllines which express cytosine deaminase activity in vitro. For 3T3-CD,PA-CD-A and PA-CD-B, the colony counts in G418 were generally greaterthan two-thirds of those in medium only controls indicating considerableenrichment of the population with cells that had incorporated plasmidand expressed enough neomycin phosphotransferase to survive in G418 1mg/ml. In 5FC alone the relative colony counts were 3-15% control countsindicating that the vast majority of the transfected cells weresensitive to 5FC. 5FC did not reduce the cloning efficiency of 3T3 orPA317 cells and thus the effect could not be attributed to the inherenttoxicity of 5FC to mammalian cells. In medium containing both 5FC andG418 almost no colonies of the cytosine deaminase expressing lines werefound.

                  TABLE 2                                                         ______________________________________                                        Relative cloning efficiency in G418 and/or                                    5FC of cell lines containing the cytosine deaminase gene.                     Cell (1)   Medium    Ave. colony number (sem)                                 line       additive  Exp. 1     Exp. 2                                        ______________________________________                                        3T3        none      171 ± 8.7                                                                             153 ± 10.4                                            G (3)      0 ± 0   0 ± 0                                                F (4)     160 ± 4.7                                                                             138 ± 6.8                                             G + F      0 ± 0   0 ± 0                                     3T3-CD     none      131 ± 4/6                                                                             130 ± 1.7                                             G         123 ± 3.5                                                                             100 ± 6.8                                             F          14 ± 1.4*                                                                             17 ± 1.4*                                            G + F      0 ± 0*  0 ± 0*                                    PA317      none       84 ± 8.1                                                                              33 ± 2.3                                             G          0 ± 0   0 ± 0                                                F          80 ± 2.5                                                                              40 ± 1.3                                             G + F      0 ± 0   0 ± 0                                     PA-CD-A    none      187 ± 5.8                                                                             137 ± 0.9                                             G         123 ± 7.7                                                                              77 ± 1.2                                             F          3 ± 1.4*                                                                              5 ± 1.5*                                             G + F      0 ± 0*  0 ± 0*                                    PA-CD-B    none      161 ± 6.0                                                                              80 ± 2.9                                             G         112 ± 7.9                                                                              38 ± 3.9                                             F          11 ± 1.7*                                                                             15 ± 1.2*                                            G + F      0 ± 0*  0 ± o*                                    3T3-CD-V1  none      143 ± 1.5                                                        G         127 ± 4.9                                                        F          9 ± 0.3*                                                        G + F      1 ± 0.3*                                             3T3-CD-V2  none      154 ± 5.2                                                        G         135 ± 4.9                                                        F          4 ± 1.2*                                                        G + F      3 ± 0.7*                                             ______________________________________                                         (1) Triplicates of each conditions were performed in each experiment.         (2) G is G418 1 mg/ml.                                                        (3) F is 5FC at 0.5 mg/ml.                                                    * P < 0.01 in comparison of (G + F) vs. (G), or (F) vs (--).             

5FC also inhibited in vitro proliferation as measured by 3H-thymidineincorporation assays. Table 3 demonstrates nearly complete inhibition of3H-thymidine incorporation by 5FC in lines 3T3-CD and PA-CD-A at aconcentration of 0.5 mg/ml without corresponding effects in the controllines 3T3 and PA317.

                  TABLE 3                                                         ______________________________________                                        Inhibition of .sup.3 H-thymidine uptake by 5FC in lines                       expressing the cytosine deaminase gene.                                                                        % medium                                     Cell    Medium.sup.(1)                                                                          Ave. cpm + sem.sup.(2)                                                                       alone.sup.(3)                                ______________________________________                                        3T3     --        35177 ± 1665                                                                              --                                                   G          120 ± 14   0.3                                                  F         50160 ± 1697                                                                              142                                                  G + F       92 ± 13   0.3                                                  5FU        880 ± 95*  2.5                                          3T3-CD  --        15520 ± 1857                                                                              --                                                   G         15283 ± 2466                                                                              98.5                                                 F          4425 ± 1182*                                                                             28.5                                                 G + F      1220 ± 409*                                                                              7.9                                                  5FU        361 ± 33*  2.3                                          PA317   --        28779 ± 1560                                                                              --                                                   G          175 ± 23   0.6                                                  F         21287 ± 1456*                                                                             74.0                                                 G + F      120 ± 14*  0.4                                                  5FU        493 ± 51*  1.7                                          PA-CD-A --        34813 ± 2791                                                                              --                                                   G         12416 ± 693 35.7                                                 F          250 ± 14*  0.7                                                  G + F      178 ± 19*  0.5                                                  5FU        263 ± 25*  0.8                                          ______________________________________                                         .sup.(1) medium was D10 alone (--), or with G418 1 mg/ml (G), 5FC 0.5         mg/ml (F), or both G418 and 5FC (G + G).                                      .sup.(2) Wells were pulsed at 72 hours with radiolabelled thymidine and       harvested 4 hours later. Values represent 12 replicates.                      .sup.(3) % medium alone = (cpm with medium additive)/(cpm control medium)     *P < 0.01 in comparison of (G + F) vs. (G), (F) vs. (--), or (5FU) vs.        (--).                                                                    

Table 4 summarizes the dose response relationship between 5FCconcentration and inhibition of cell line PA-CD-A in both clonogenic andproliferation assays. In line PA-CD-A, 5FC profoundly inhibited both ³H-thymidine incorporation and colony counts over a concentration rangeof 62-500 μg/ml; below that range the effects were diminished somewhatbut still significant. No corresponding inhibition of the control PA317cells were observed.

                  TABLE 4                                                         ______________________________________                                        5FC selectively eliminates cells expressing the                               cytosine deaminase gene from mixed population in vitro.                                     Average colony count ± sem.sup.(3)                           Cells.sup.(1)                                                                          Medium.sup.(2)                                                                           Exp. 1      Exp. 2                                        ______________________________________                                        PA-CD-A  --          122 ± 5.6                                                                             126 ± 1.5                                           G            90 ± 4.5                                                                              85 ± 2.3                                           F            7 ± 0.6                                                                               14 ± 3.0                                           G + F       0.7 ± 0.3                                                                              1 ± 0.0                                   PA317    --           83 ± 2.0                                                                              74 ± 1.5                                           G            0 ± 0   0 ± 0                                              F            85 ± 3.0                                                                              71 ± 5.7                                           G + F        0 ± 0   0 ± 0                                     PA-CD-A  --          164 ± 5.5                                                                             140 ± 5.6                                  +        G            83 ± 2.7                                                                              72 ± 3.2                                  PA317    F            84 ± 1.5                                                                              72 ± 1.5                                           G + F       0.7 ± 0.3                                                                              1 ± 0.3                                   ______________________________________                                         .sup.(1) 10.sup.3 PACD-A, 10.sup.3 PA317, or 10.sup.3 PACD-A plus 10.sup.     PA317 cells were inoculated into wells.                                       .sup.(2) Medium was D10 with the following additives: (--) none, (G) G418     1 mg/ml, (F) 5FC 125 μg/ml, (G + F) G418 1 mg/ml and 5FC 125 μg/ml.     .sup.(3) Values represent average and sem of 3 replicates.               

Table 5 demonstrates the selectivity of 5FC toxicity in mixed cellpopulations. Equal numbers of PA-CD-A cells (which contain the cytosinedeaminase gene) and PA317 cells (which do not) were mixed and inoculatedinto wells in clonogenic assays and subjected to selection in 5FC, G418or both. If the 5FC toxicity were restricted to cells containing thecytosine deaminase gene, one would expect approximately one-half thecells to be eliminated from the mixed population, while nearly all thecells in a pure PA-CD-A population and none of the cells in a pure PA317population would be affected by 5FC. However if there were significant"bystander killing" by release of cytosine deaminase and/or 5-FU intothe medium one would expect both PA-CD-A and PA317 cells in the mixedpopulation to be killed. The colony count in 5FC was approximatelyone-half the colony count of the mixed population in nonselective media,while control PA317 cells were unaffected by 5FC and PA-CD-A cells werekilled.

Finally, the presence and expression of the cytosine deaminase gene hadno deleterious effects on cells in the absence of 5FC as judged bycloning efficiency, proliferation in vitro, growth rates in culture ormicroscopic morphology.

                  TABLE 5                                                         ______________________________________                                        5FC selectively eliminates cells expressing the                               cytosine deaminase gene from mixed populations in vitro.                                    Average colony count ± sem.sup.(3)                           Cells.sup.(1)                                                                          Medium.sup.(2)                                                                           Exp. 1      Exp. 2                                        ______________________________________                                        PA-CD-A  --          122 ± 5.6                                                                             126 ± 1.5                                           G            90 ± 4.5                                                                              85 ± 2.3                                           F            7 ± 0.6                                                                               14 ± 3.0                                           G + F       0.7 ± 0.3                                                                              1 ± 0.0                                   PA317    --           83 ± 2.0                                                                              74 ± 1.5                                           G            0 ± 0   0 ± 0                                              F            85 ± 3.0                                                                              71 ± 5.7                                           G + F        0 ± 0   0 ± 0                                     PA-CD-A  --          164 ± 5.5                                                                             140 ± 5.6                                           G            83 ± 2.7                                                                              72 ± 3.2                                           F            84 ± 1.5*                                                                             72 ± 1.5*                                          G + F       0.7 ± 0.3                                                                              1 ± 0.3                                   ______________________________________                                         .sup.(1) 10.sup.3 PACD-A, 10.sup.3 PA317, or 10.sup.3 PACD-A plus 10.sup.     PA317 cells were inoculated into wells.                                       .sup.(2) Medium was D10 with the following additives: (--) nonc, (G) G418     1 mg/ml, (F) 5FC 125 μg/ml, (G + F) G418 1 mg/ml and 5FC 125 μl/ml.     .sup.(3) Values represent average and sem of 3 replicates.                    *Values do not differ significantly from PA317 colony counts in unmodifie     medium or medium containing 5FC.                                         

EXAMPLE 4 Retrovirus Mediated Gene Transfer Results in SuccessfulExpression of the Cytosine Deaminase Gene

3T3 cells were transduced by exposure to the supernatant from cell linesPA-CD-A and PA-CD-B and selected in G418 as described above in thematerials and methods section. The resultant cell lines were designated3T3-CD-V1 (transduced with retrovirus from PA-CD-B) and 3T3-CD-V2(transduced with retrovirus from PA-CD-A). As seen in Table 1 lysates ofboth 3T3-CD-V1 and 3T3-CD-V2 converted cytosine to uracil in vitro. Theywere also sensitive to 5FC in clonogenic assays (Table 2).

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 109 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAATTCGTTAACGCGGTATTAGGTGGCGCGCTGAGCTATCTGATCCTTAACCCGATTTTG60                AATCGTAAAACGACAGCAGCAATGTCGCATGTGGAGGCTAACAGTGTCG109                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GAATTCAGGCTAGCAATGTCG21                                                       (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TGACGCGAATTCAGGCTAGCAATGTCG27                                                 (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CACACATTCCACAGCGGATCC21                                                       (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CAATGTCGCATGTGGAGGCTAACAGTGTCG30                                              __________________________________________________________________________

What is claimed is:
 1. A method for selectivity killing mammalian hostcells in vitro, comprising the steps of:(a) inserting an E. colicytosine deaminase gene into a mammalian expression vector, saidcytosine deaminase gene modified at the GTG translation initiation codonto ATG to facilitate initiation of translation in mammalian cells, (b)inserting an exogenous DNA of interest into said mammalian expressionvector, (c) introducing the mammalian expression vector comprising thecytosine deaminase gene and the exogenous DNA of interest into mammalianhost cells in vitro, such that the mammalian expression vectorintegrates into the genome of the mammalian host cells, and (d) treatingthe mammalian host cells of (c) with 5-fluorocytosine, wherein thosemammalian host cells expressing the E. coli cytosine deaminase gene willbe selectively killed.
 2. The method of claim 1, wherein said exogenousDNA of interest is a gene of interest.
 3. A method for selectivelykilling mammalian host cells in vitro, comprising the steps of:(a)inserting an E. coli cytosine deaminase gene into a first mammalianexpression vector, said cytosine deaminase gene modified at the GTGtranslation initiation codon to ATG to facilitate initiation oftranslation in mammalian cells, wherein said first mammalian expressionvector also contains a gene which encodes neomycin resistance, (b)introducing said first mammalian expression vector comprising thecytosine deaminase gene and the gene which encodes neomycin resistanceinto mammalian host cells in vitro, such that said first mammalianexpression vector integrates into the genome of the mammalian hostcells, (c) treating the mammalian host cells of (b) with neomycin toenrich the population of treated cells with cells that have taken up andexpress genes present in the first mammalian expression vector, (d)introducing a second mammalian expression vector comprising an exogenousDNA of interest into the mammalian host cells of (c) such that saidsecond mammalian expression vector integrates into the genome of themammalian host cells of (c), and (e) treating the mammalian host cellsof (d) with 5-fluorocytosine, wherein those mammalian host cellsexpressing the E. coli cytosine deaminase gene will be selectivelykilled.
 4. The methods of claim 3, wherein said exogeneous DNA ofinterest is a gene of interest.
 5. A method for rendering mammalian hostcells susceptible to 5-fluorocytosine, comprising introducing intomammalian host cells in vitro an expression vector that contains acytosine deaminase gene modified at the GTG translation initiation codonto ATG to facilitate initiation of translation in mammalian host cells,wherein the modified cytosine deaminase gene is expressed in themammalian host cells, thereby rendering the mammalian host cellssusceptible to 5-fluorocytosine.
 6. The methods of claim 5, wherein saidexpression vector further comprises an exogenous DNA of interest.